EP2251692B1 - Procédé de classification d'échantillons des patients atteints par la sclérose en plaques - Google Patents

Procédé de classification d'échantillons des patients atteints par la sclérose en plaques Download PDF

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EP2251692B1
EP2251692B1 EP20100169971 EP10169971A EP2251692B1 EP 2251692 B1 EP2251692 B1 EP 2251692B1 EP 20100169971 EP20100169971 EP 20100169971 EP 10169971 A EP10169971 A EP 10169971A EP 2251692 B1 EP2251692 B1 EP 2251692B1
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interferon
type
genes
ifn
individual
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EP2251692A1 (fr
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Elisabeth Geertruida Maria Baarsen Van
Saskia Stahlecker-Voslamber
Cornelis Lammert Verweij
Chris Hubert Polman
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Vereniging voor Christelijik Hoger Onderwijs Wetenschappelijk Onderzoek en Patientenzorg
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Interferons are natural proteins produced by the cells of the immune system in response to challenges by foreign agents such as viruses, bacteria, parasites and tumor cells.
  • Today, interferons are approved for treatment of malignancies such as hairy cell leukemia, malignant melanoma, and AIDS-related Kaposi's sarcoma; chronic hepatitis B and C; multiple sclerosis; condylomata acuminate, genital and perianal warts caused by infections with human papillomavirus (HPV); chronic granulomatous disease; renal cell carcinoma (RRC) and severe, malignant osteopetrosis.
  • Clinical trials are ongoing or have been finished to show a clinical benefit of interferon-comprising treatment for other malignancies, virus-mediated diseases and autoimmune-related diseases such as rheumatoid arthritis.
  • MS Multiple sclerosis
  • IFNs were the first agents to show clinical efficacy in Relapsing Remitting MS (RRMS).
  • Interferon beta IFNbeta
  • IFNbeta Interferon beta
  • therapy is associated with a number of adverse reactions, including flu-like symptoms and transient laboratory abnormalities.
  • response to IFNbeta is partial, i.e. disease activity is suppressed by only about one third (2).
  • Clinical experience suggests that there are IFN 'responders' as well as 'non responders' (3;4). In the absence of predictive biomarkers the question remains who will respond to therapy and who to treat when inconvenience and costs are significant.
  • IFNs In normal physiology IFNs produce their biological effects by binding to multi-subunit receptors IFNAR-1 and -2 on the cell surface, thereby initiating a complex cascade of intracellular secondary messengers that emerge in two divergent pathways.
  • One pathway leads to activation of the transcription factor ISGF3 (IFN-stimulated gene factor 3), a complex of phosphorylated Signal Transducer and Activator of Transcription (STAT) 2 with STAT1 and IFN regulatory factor 9 (IRF-9; p48) that binds to the IFN-stimulated response element (ISRE) present in multiple genes (9;10).
  • ISGF3 IFN-stimulated gene factor 3
  • STAT1 phosphorylated Signal Transducer and Activator of Transcription
  • IRF-9 IFN regulatory factor 9
  • the other pathway involves STAT2/1 and STAT2/3 heterodimers and STAT1 homodimer (IFNalpha-activated factor, AAF), which bind to the IFN gamma-activated sequence (GAS) response element (10-13).
  • AAF IFNalpha-activated factor
  • GAS IFN gamma-activated sequence
  • the present disclosure provides a method for classifying cells from a human individual said method comprising providing a sample comprising cells from said individual that are typically responsive to exposure to a type I interferon, determining a level of activity of a pathway that is modulated by type 1 interferon, and classifying said cells on the basis of the determined level of activity. It is preferred that said pathway comprises an activation pathway, whereby said activation pathway comprises the transcriptional activation of genes.
  • An important application of the present invention is to determine whether treatment of said individual with a type I interferon is likely to be successful. An individual that has cells that are classified as poor responders is likely not respond well to treatment with a type I interferon.
  • a preferred method further comprises culturing said cells in the presence of a type I interferon prior to determining a level of activity of said pathway.
  • said individual suffers from, or is at risk of suffering from, an autoimmune disease.
  • autoimmune disease refers to a disease that is characterized by an immune response against an antigen that is normally present in the body, or that mimics a substance that is normally present in the body.
  • Typical autoimmune diseases comprise multiple sclerosis (MS), Crohn's disease and rheumatoid arthritis.
  • malignancies such as hairy cell leukemia, malignant melanoma, and AIDS-related Kaposi's sarcoma; chronic hepatitis B and C; multiple sclerosis; condylomata acuminate, genital and perianal warts caused by infections with human papillomavirus (HPV); chronic granulomatous disease, severe, malignant osteopetrosis, chronic viral hepatitis, heamatological malignancies such as multiple myeloma, and renal cell carcinoma.
  • Chronical viral hepatitis, heamatoligical malignancies and renal cell carcinomas are treated with IFNalpha, while MS and multiple myeloma are treated with IFNbeta.
  • a preferred autoimmune disease for application of a method of the invention is multiple sclerosis (MS), more preferred Relapsing Remitting MS.
  • the responsiveness of cells that are typically responsive to exposure to a type I interferon significantly differs prior to treatment. This difference is indicative for the responsiveness of the individual to treatment with said type I interferon, and can be determined by determining a level of activity of a pathway that is modulated by type 1 interferon.
  • the samples are preferably classified as being derived from an individual with a high, low or intermediate probability of being non-responsive or responsive to treatment with said type I interferon, based on the determined level of activity.
  • a response to exposure to a type I interferon can be determined clinically.
  • Several criteria are known in the art (3;4) and can be used for determining a clinical response.
  • Preferred criteria are determining a change in Expanded Disability Status Score (EDSS); a difference in relapse rate 2 years before and 2 years after start of the therapy; and/or formation of a new T2 lesion as measured by magnetic resonance imaging.
  • Non-responsiveness is preferably determined by an increased disability as determined by an increase in EDSS after 6 months of exposure to interferon and/or the presence of one or more relapses during exposure to interferon.
  • interferon alpha interferon alpha
  • interferon beta interferon beta
  • a completely artificial type I interferon was generated from the amino acid composition of interferon alpha and beta.
  • This molecule was termed "consensus" interferon.
  • a molecule is said to be a type I interferon if it is interferon alpha, interferon beta or a functional part, derivative and/or analogue thereof having at least the same activity in kind as a type I interferon although the amount of activity does not necessarily need to be the same.
  • a functional part of IFN is a part of IFN comprising the same gene activity modulating activity in kind as IFN itself.
  • the amount of activity of such a part may differ from the activity of the complete protein.
  • a person skilled in the art is capable of generating a suitable derivative of IFN. Derivatives can, for instance, be obtained by conservative amino acid substitution, indeed some of the currently prescribed human interferons differ slightly in amino acid sequence from natural human interferons.
  • type I interferons examples include: Rebif TM , a liquid form of Interferon beta 1a; Avonex TM , lyophilized form of Interferon beta 1a; Cinnovex TM , generic/biosimilar form of Interferon beta 1a (Avonex TM ); Betaseron TM , Interferon beta 1 b; Roferon A TM , regular Interferon-alpha2a; Intron-A TM , regular Interferon-alpha2b; and Pegasys TM , Pegylated Interferon alpha 2a.
  • a type I interferon of the invention may thus also be modified chemically, for instance through the addition of PEG.
  • a suitable part of IFN is for instance a part with an altered glycosylation pattern or a part that is non-glycosylated. Glycosylation can be prevented by removing or altering a glycosylation site of the molecule. If the generation of such a (partially) deglycosylated IFN requires alteration of the amino acid composition than such a deglycosylated IFN is derivative of a functional part of IFN.
  • a functional part, derivative and/or analogue of IFN comprises the same activity in kind not necessarily in amount.
  • IFN may modulate the profile of cytokine production toward that of the antiinflammatory phenotype (for instance by upregulation of IL-10), and this appears to occur in the systemic circulation and within the CNS.
  • All type-I interferons exert their effect through the type-I-interferon-receptor (IFN-R1).
  • IFN-R1 type-I-interferon-receptor
  • a functional part, derivative and/or analogue of IFN therefore preferably comprises the same signalling activity through IFN-R1 in kind not necessarily in amount.
  • interferon-beta was used to demonstrate the effectiveness of a method of the invention, however, an alternative type I interferon (for example, interferon-alpha) is also effective.
  • a type I interferon has activity on human cells. Both human and primate IFN is active in humans.
  • said type I interferon is a primate type I interferon or a functional part, derivative and/or analogue thereof.
  • said type I interferon is a human type I interferon.
  • said type I interferon is interferon beta or a functional part, derivative, analogue and/or equivalent thereof.
  • a type I interferon many cells are responsive for a type I interferon.
  • cells that are typically responsive to exposure to a type Interferon are meant cells of a type that, when obtained from normal (healthy) individuals, are responsive to a type I interferon, when exposed thereto in a normal amount/concentration.
  • Non-limiting examples of such cells comprise cheek cells as present in buccal mucosal scrapings, and epithelial cells such as keratinocytes.
  • a preferred example of a sample of cells is a sample of blood, or total blood cells.
  • the sample comprises peripheral blood mononuclear cells (PBMC) or a cell fraction thereof that is typically responsive to exposure to a type I interferon.
  • PBMC peripheral blood mononuclear cells
  • Particularly preferred examples of such cells are peripheral monocytes, B cells and T cells.
  • a method of the disclosure is particularly suited for the classification of samples or cells of individuals suffering from or at risk of suffering from multiple sclerosis.
  • the responsiveness of an individual suffering from or at risk of suffering from multiple sclerosis towards type 1 interferon-mediated treatment is inversely related to the basal level of activity of a pathway that is modulated by type 1 interferon prior to said treatment.
  • An increased activity of said pathway prior to treatment reduces the chance of responding to said treatment, whereby said activity is compared to the activity of reference sample comprising cells from, for example, an individual not suffering from multiple sclerosis, or an individual suffering from multiple sclerosis but being responsive to said treatment.
  • a threshold can be set, based on data obtained from responders and non-responders. If the basal level of activity of a pathway scores above said threshold, said responsiveness can be classified as having an increased risk of being non-responsive. If the basal activity score below said threshold, said responsiveness can be classified as having an increased risk of being responsive. It will be clear to a skilled person that more than one arbitrary threshold can be set for classifying cells from a human individual depending on the derived reliability of the classification.
  • a level of activity of a pathway that is modulated by type 1 interferon can be measured in a number of ways known in the art.
  • Known methods comprise determining a cellular localization or a level of phosphorylation of intermediate signalling molecules such as STAT1, STAT2 and IRF7 with antibodies and for example confocal microscopy or fluorescence-activated cell sorting, and determining serum levels of interferon-regulated cytokines such as for example, interleukin 6 and 15, CCL2 (MCP-1), CCL3, CCL8 (MCP-2), CCL19, and CXCL9, 10, and 11 using multiplex immunoassay.
  • said level of activity is determined by determining an expression level of at least one gene of table 2, LGALS3BP or Siglec-1 in said cells.
  • the genes listed in table 2, LGALS3BP and Siglec-1 were found to be indicative of a level of activity of a pathway that is modulated by type 1 interferon, and, therefore, can be used for determining said activity.
  • a preferred method for determining an expression level is determining a level of RNA in said cells.
  • the level of RNA in said cells is determined for at least one gene of table 2, LGALS3BP or Siglec-1.
  • said at least one gene that is listed in table 2 LGALS3BP or Siglec-1 has an R value of at least -0.65.
  • the RNA levels in said cells of at least 5 and preferably at least 10, more preferably at least 15 genes of table 2 are determined.
  • said at least 5, 10 or 15 genes each comprise an R value of at least -0.65 in said table.
  • RNA levels of less than 5 genes it is preferred that at least one of the determined RNA levels involves the level of RSAD2.
  • RNA levels of at least 5 genes it is preferred that the RNA level of at least RSAD2 (Viperin), IFIT1 (alias G10P1; IF156; RNM561), MX1 (alias MxA; IF178), G1P2 (alias ISG15; IFI15), and Image: 1926927 are determined.
  • At least the genes or the genes products of the genes RSAD2, IFIT1, MX1, ISG15, EPSTI1, IRF7, LY6E, OAS1, OAS3, SERPING1 are measured. These provide an even better result than the already suitable first 5 of table 2.
  • at least the first 10 genes and or gene products of table 2 are measured (i.e. RSAD2 till LY6E). These also provide a better result than the already suitable first 5 of table 2.
  • said sample is a sample that has been obtained from said individual prior to initiation of the treatment with said type I interferon.
  • the gene signatures can also be used to follow and/or determine the responsiveness of the individual once the treatment has been initiated.
  • said sample comprises a sample that is obtained from the individual both before and after initiation of the treatment with said type I interferon.
  • the classification is preferably done by comparing the determined level of activity with a reference.
  • the reference can be a determined level of activity of said pathway from another sample of cells of said individual.
  • one of said sample is collected prior to initiation of treatment with a type I interferon and another of said sample is collected after initiation of said treatment.
  • a method further comprises comparing said level of activity with the level of activity of said pathway in a sample of cells from said individual while receiving treatment with a type I interferon.
  • cells in a sample are divided into two fractions wherein the level of activity of said pathway is determined for cells of both of said fractions and wherein a first of said fractions comprising untreated cells (resting cells) and wherein a second of said fractions comprises cells that have been cultured in the presence of a type I interferon prior to determining a level of activity of said pathway, said method further comprising classifying said cells on the basis of a comparison of the level of activity in said two fractions.
  • the untreated cells may be cells that are directly frozen after collection.
  • the untreated cells may also be separated from serum and/or other cells in the collection sample prior to freezing.
  • the untreated cells may also be a whole blood sample.
  • the cells may be cultured, however, to remain "untreated" they may not be cultured together with a type I interferon.
  • the untreated sample is a protein sample it is preferred that the untreated sample is a serum sample.
  • expression levels are compared between resting and type I interferon (preferably IFNbeta) treated purified PBMC.
  • PBMC preferably IFNbeta
  • said PBMC are derived from the same sample wherein one part of said sample represents resting PBMC.
  • Another part of said sample is cultured according to the invention in the presence of a type I interferon (preferably IFNbeta) and represents type I interferon (preferably IFNbeta) treated purified PBMC.
  • said sample is a sample from an individual that is not treated with interferon at the time of sample collection.
  • said individual is an individual that is being prescreened for type I interferon responsiveness, preferably for determining whether the individual is to be treated for MS with a type I interferon.
  • the expression levels for the comparison are determined for the genes RSAD2, MxA and STAT1.
  • said expression levels are determined by means of quantitative PCR, preferably by means of quantitative real-time PCR.
  • Expression profiles of sets of genes can be determined using a variety of methods. Methods for determining RNA expression level, such as Northern blotting, are known in the art and can be applied for the current invention. Preferred examples are quantitative amplification methods such as PCR, and methods involving the use of (micro)arrays containing probes for the respective RNAs. Preferred PCR-based methods comprise multiplex PCR and multiplex ligation-dependent probe amplification. The array format is particularly useful for this purpose. Using an array format it is possible to generate gene signatures that discriminate between individuals that have a high, low or intermediate probability to be responsive to treatment with a type I interferon.
  • Microarrays consist of solid support on which DNA fragments derived from individual genes are placed in an ordered array. These arrays are hybridized with fluorescent cDNA probes prepared from cellular mRNA.
  • Two types of microarrays are most commonly used. One comprises oligonucleotides that are produced by in situ oligonucleotide synthesis using photolithographic masking techniques. In this type, genes are represented by 11 to 16 oligonucleotides (25-mers), each including a perfect match and a mismatch that is identical except for a single base mismatch in its center.
  • Another type of microarrays consists of longer sequences (20-2000 bp) of cDNA (PCR products) or oligonucleotides with each element representing a distinct gene that are printed on glass microscope slides (15-16).
  • Hybridization of the cDNA probes to microarrays results in specific base pairing with the corresponding gene sequence at known locations on the microarray.
  • the specific hybridization signal of the fluorescent cDNA probes to each DNA spot is quantified using a confocal scanning device.
  • the scanned images are transformed into a gene expression matrix.
  • different bioinformatics software can be applied to analyze the data. Data analysis comprises normalization of the data to reduce bias within and between experiments.
  • a preferred method for determining an RNA expression level comprises Taqman Low Density Arrays (TLDA; Applied Biosystems), which are pre-loaded customizable 384-well micro fluidic cards for target class and pathway studies based on Taqman realtime PCR. Custom-designed TLDA cards can be used to measure genes of interest. Using this high-throughput system the expression of all genes can be analyzed simultaneously for up to eight samples using minimal amounts of sample.
  • TLDA Taqman Low Density Arrays
  • An alternative method for determining an expression level of at least one gene of table 2 LGALS3BP or Siglec-1 is by determining a protein expression level.
  • Said protein expression level can be determined by any method known to a skilled person, including but not limited to Western blotting, flow cytometry, immunohistochemistry, and enzyme-linked immuno sorbent assay (ELISA).
  • Preferred methods comprise flow cytometry and/or ELISA.
  • the disclosure further provides a kit of part comprising a set of probes or primers specific for RNA of at least 5 and preferably at least 10, more preferably at least 15 genes of table 2.
  • a kit of part comprising a set of probes or primers specific for RNA of at least 5 and preferably at least 10, more preferably at least 15 genes of table 2.
  • said at least 5, 10 or 15 genes each comprise an R value of at least -0.65 in said table.
  • the disclosure further provides the use of a kit according to the invention for classifying a sample of an individual suffering from or at risk of suffering from multiple sclerosis.
  • the disclosure provides a method for classifying an individual as an individual with a reduced capacity to respond to type 1 beta interferon mediated therapy, said method comprising:
  • the TT genotype of rs2004640 is associated with low/bad biological response.
  • the TT genotype of rs2004640 is associated with low/bad clinical response.
  • the AA genotype of rs4728142 is associated with low/bad biological response.
  • the AA genotype of rs4728142 is associated with low/bad clinical response.
  • the TT genotype of rs2004640 is associated with high baseline levels of the IFN response genes of the gene set.
  • the AA genotype of rs4728142 is associated with high baseline levels of the IFN response genes of the gene set. Patients homozygous for the 5bp CGGGG deletion show a low/bad biological response. Patients homozygous for the 5bp CGGGG deletion show a bad clinical response.
  • more than one polymorphism is determined to indicate whether an individual is a good or bad responder to treatment with a type I interferon. It is preferred to determine the haplotype for the indicated polymorphisms and classify the cells of the individual on the basis of of the determined haplotype.
  • Haplotype 1 rs4728142 (A) rs2004640 (T) exon 6 indel (del) rs10954213 (A)
  • Haplotype 8 rs4728142 (G) rs2004640 (G) exon 6 indel (in) rs10954213 (G)
  • Haplotype 1 rs4728142 (A) rs2004640 (T) exon 6 indel (del) rs10954213 (A)
  • Haplotype 8 rs4728142 (G) rs2004640 (G) exon 6 indel (in) rs10954213 (G)
  • the invention provides the use a polymorphism as indicated in figure 10 for determining whether an individual is likely to be a good, bad or normal responder to treatment with a type I interferon.
  • Said polymorphism is a polymorphism of rs4728142, rs2004640, exon 6 indel (del) or exon 6 indel (in).
  • the haplotype for at least two and preferably at least 3 and more preferably all of the polymorphisms mentioned are determined.
  • the polymorphism is also determined according to the invention if the complementary strand is analysed. In this case, the correlations and predictions as indicated herein above, are of course associated with the presence of the respective complementary nucleotide(s).
  • the present invention further provides a method for classifying an individual as an individual with a reduced capacity to respond to type 1 interferon mediated therapy, said method comprising:
  • Example 1 see also: van Baarsen LG, Vosslamber et al., PLoS ONE. 2008)
  • PBMCs peripheral blood mononuclear cells
  • RNA was isolated using the PreAnalytix RNA isolation kit according to the manufacturers' instructions, including a DNAse (Qiagen) step to remove genomic DNA. Quantity and quality of the RNA was tested using the Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, Delaware USA)o the manufacturers' instructions, including a DNAse (Qiagen) step to remove genomic DNA. Quantity and quality of the RNA was tested using the Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, Delaware USA).
  • RNA (0.5 ⁇ g) was reverse transcribed into cDNA using a Revertaid H-minus cDNA synthesis kit (MBI Fermentas, St. Leon-Rot, Germany) according to the manufacturers' instructions. Quatative realtime PCR was performed using an ABI Prism 7900HT Sequence detection system (Applied Biosystems, Foster City, CA, USA) using SybrGreen (Applied Biosystems). Primers were designed using Primer Express software and guidelines (Applied Biosystems) and are listed in table 4. To calculate arbitrary values of mRNA levels and to correct for differences in primer efficiencies a standard curve was constructed. Expression levels of target genes were standardized against housekeeping gene glyceraldehydes-3-phosphate dehydrogenase (GAPDH), parallel detected in the identical cDNA samples.
  • GPDH housekeeping gene glyceraldehydes-3-phosphate dehydrogenase
  • PBMCs Freshly isolated PBMCs were washed using PBS containing 1% fetal calf serum (FCS; BioWhittaker, Cambrex) and plated in 24-wells culture plates at a density of 2x106 cells per ml per well. Cells were stimulated or not with 10 Units recombinant IFNbeta (Abcam, Cambridge, UK) for 4h after which RNA was isolated using the Rneasy Qiagen RNA isolation kit (Qiagen, Venlo, Netherlands) according to the manufacturers' instructions. A DNAse (Qiagen) step was included to remove genomic DNA. Quantity and quality of the RNA was tested using the Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, Delaware USA)
  • Correlation analyses were performed using Graphpad Prism 4 software. First, data was tested for normal distribution. In case data passes normality test, correlation was tested using Pearson correlation. Spearman correlation was used in case of nonparametric distribution of the data. Correlation was considered significant if p-values were less than 0,05.
  • Example 2 see also: van Baarsen LG, Vosslamber et al., PLoS ONE. 2008)
  • Example 3 see also: van Baarsen LG, Vosslamber et al., PLoS ONE. 2008)
  • Example 4 see also: van Baarsen LG, Vosslamber et al., PLoS ONE. 2008)
  • the selected IFNbetaresponse genes are i, RSAD2, which shows the most significant correlation of biological response versus baseline at single gene level (Table 2); ii, MxA, which shows a good negative correlation and is known as a marker of IFN bioactivity (23); and iii, STAT1, which is one of the components that is important for IFNbetasignaling.
  • ii, MxA which shows a good negative correlation and is known as a marker of IFN bioactivity
  • STAT1 which is one of the components that is important for IFNbetasignaling.
  • Example 5 see also: van Baarsen LG, Vosslamber et al., PLoS ONE. 2008)
  • gene expression levels of RSAD2 (among others) may predict the biological response to IFNbeta treatment. In this second group of patients we measured the expression of RSAD2 using quantitative realtime PCR.
  • peripheral blood was collected from 30 RRMS patients before and during IFN-b therapy. From 20 untreated RRMS patients peripheral blood was collected at two time points over a three to twelve month time period to analyze the stability of baseline values over time. Baseline stability and biological response rate, were analyzed by Taqman Low Density Arrays (TLDA) using the mean gene expression level of a set of the top 10 IFN type I response genes of table 2 (RSAD2, IFIT1, MX1, ISG15, EPSTI1, IRF7, LY6E, OAS1, OAS3, SERPING1). Genetic variation was determined the IRF5 gene, a component of the IFN signaling cascade.
  • TLDA Taqman Low Density Arrays
  • the SNPs were genotyped using Taqman Genotyping Assay (Applied Biosystems).
  • the 30bp indel was amplified as a 115/145 bp fragment using conventional PCR.
  • the PCR-amplified fragments were separated on a 2.5% agarose gel and visualized using ethidium bromide staining.
  • haplotype 1 is associated with bad/low biological response and haplotype 8 is associated with good/high biological response.
  • LGALS3BP alias protein 90K/Mac-2BP gene expression in whole blood from 15 MS patients before and after IFN beta therapy
  • gene expression levels of LGALS3BP (among others) may predict the biological response to IFNbeta treatment.
  • LGALS3BP In patients with a high expression of LGALS3BP before start of the treatment, the expression level is low after/during treatment whereas in the patients with low expression of LGALS3BP before treatment the expression clearly raises after/during IFN beta treatment (see Figure 8 and Figure 9 ). Therefore, elevated serum levels of LGALS3BP are a predictive biomarker for failure to respond to type I IFN treatment, as has been shown previously for interferon-alpha treated patients with chronic hepatitis C infection ( Artini M et al., Hepatol. 1996 Aug;25(2):212-7 ).
  • Sialic acid-binding Ig-like lectin 1 (Siglec-1, sialoadhesin, CD169)) is known as one of the most prominent type I IFN-regulated candidate genes. Biesen et al. (Arthr. Rheum. 2008 Apr;58(4):1136-45 ) demonstrated that Siglec-1 expression in inflammatory and resident monocytes is a potential biomarker for monitoring disease activity and success of therapy in systemic lupus erythematosus. Levels of Siglec-1 were determined using multicolor flow cytometry. It was shown that the frequency of Siglec-1-expressing monocyte subsets was correlated with disease activity (as measured by the SLE Disease Activity Index) and was inversely correlated with levels of complement factors.
  • anti-dsDNA anti-double-stranded DNA
  • glucocorticoid treatment resulted in a dramatic reduction of Siglec-1 expression in cells from patients with active SLE.
  • Siglec-1 expression in resident blood monocytes is a potential biomarker for type I IFN responses that are indicative for disease activity. Therefore, levels of Siglec-1 expressed on monocytes in MS and other IFN related diseases (melanona, Hepatitis C infection) can be used as a biomarker for baseline type I IFN response avtivity, that is predictive for the response to treatment.
  • Sense primer Antisense primer Length PCR product MxA NM 002462 TTCAGCACCTGATGGCGTATC GTACGTCTGGAGCATGAA GAACTG 92 OAS1 NM 016816 TGCGCTCAGCTTCGTACTGA GGTGGAGAACTCGCCCTCTT STAT1 NM 007315 TGC ATC ATC GGC TTC ATC AGC GAA GTC AGG TTC GCC TCC GTT C 156 RSAD2 NM 080657 CTGGTTCCAGAATTATGGTTAGTATTT CCACGGCCAATAAGGACATT 90 IRF7 NM 004031 GCTCCCCACGGTATACCATCTAC GCCAGGGTTCCAGCTTCAC 99 ISG15 NM 005101 TTTGCCAGTACAGGAGCTTGTG GGGTGATCTGCGCCTTCA 151 IFNb NM 002176 ACAGACTTACAGGTTACCTCCGAAAC CTCCTAGCCTGTCCCTCTGGGACTGG 93

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Claims (9)

  1. Procédé de classification d'un individu souffrant d'une maladie pouvant être traitée par un interféron en tant qu'individu ayant une capacité réduite à répondre à un traitement avec une thérapie médiée par un interféron de type 1, ledit procédé comprenant les étapes de :
    - détermination dans un échantillon d'acide nucléique dudit individu d'un ou plusieurs polymorphismes,
    - détermination, sur la base du génotype dudit/desdits polymorphisme(s), si l'individu a une capacité réduite, normale et/ou bonne à répondre à une thérapie médiée par un interféron de type 1,
    dans lequel les un ou plusieurs polymorphismes sont situés dans le gène IRF5 et sont choisis dans le groupe constitué des polymorphisme de nucléotide unique (SNP) rs2004640, SNP rs4728142, et polymorphisme d'insertion-délétion de 30 pb dans l'exon 6 ACTCTG[CGGCCGCCTACTCTGCAGCCGCCCACTCTG]CAGCCG
    le génotype TT de rs2004640 étant associé à une réponse biologique et clinique faible/mauvaise, le génotype AA de rs4728142 étant associé à une réponse biologique et clinique faible/mauvaise et la délétion de 30 pb dans l'exon 6 étant associée à une réponse biologique et clinique faible/mauvaise.
  2. Procédé selon la revendication 1 dans lequel ladite maladie pouvant être traitée par un interféron est une maladie auto-immune.
  3. Procédé selon la revendication 2, dans lequel ladite maladie auto-immune est la sclérose en plaques.
  4. Procédé selon la revendication 1 dans lequel ladite maladie pouvant être traitée par un interféron est choisi parmi La maladie de Crohn et la polyarthrite rhumatoïde, des malignités telles que la leucémie à tricholeucocytes, un mélanome malin, et le sarcome de Kaposi lié au SIDA ; l'hépatite B et C chronique ; la sclérose en plaques ; des condylomes acuminés, des verrues génitales et périanales causées par des infections par le virus du papillome humain (VPH) ; la granulomatose chronique, l'ostéopétrose maligne grave, l'hépatite virale chronique, des malignités hématologiques telles que le myélome multiple, et le carcinome à cellules rénales ; et l'hépatite virale chronique.
  5. Procédé selon les revendications 1 à 4, dans lequel ledit interféron de type I est l'interféron bêta.
  6. Procédé selon les revendications 1 à 5 dans lequel plusieurs polymorphismes sont déterminés.
  7. Procédé selon les revendications 1 à 6 dans lequel l'échantillon dudit individu contient des cellules qui répondent de manière typique à une exposition à un interféron de type I.
  8. Procédé selon la revendication 7 dans lequel lesdites cellules sont des cellules sanguines.
  9. Procédé selon les revendications 1 à 8 dans lequel l'échantillon a été obtenu à partir dudit individu avant le début de la thérapie médiée par un interféron de type 1.
EP20100169971 2007-06-01 2008-06-02 Procédé de classification d'échantillons des patients atteints par la sclérose en plaques Not-in-force EP2251692B1 (fr)

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EP20100169971 EP2251692B1 (fr) 2007-06-01 2008-06-02 Procédé de classification d'échantillons des patients atteints par la sclérose en plaques
EP08766766A EP2153226A2 (fr) 2007-06-01 2008-06-02 Supports et procédé de classification d'échantillons des patients atteints par la sclérose en plaques

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WO2012024236A1 (fr) * 2010-08-14 2012-02-23 Biogen Idec Ma Inc. Blocage des ltβr : procédés pour optimiser la sensibilité thérapeutique de patients
US20130261018A1 (en) * 2010-10-20 2013-10-03 University Of Tennessee Research Foundation Interferon gene signature and methods of use thereof
WO2012074396A1 (fr) * 2010-11-30 2012-06-07 Vereniging Voor Christelijk Hoger Onderwijs, Wetenschappelijk Onderzoek En Patiëntenzorg Méthode de pronostic de la réponse clinique d'un patient à une thérapie d'inhibition ou d'épuisement des stocks de lymphocytes b dans des maladies induites par l'interféron comme le lupus érythémateux aigu disséminé

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CA2689431A1 (fr) 2008-12-04
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CN101720435A (zh) 2010-06-02
JP2014121321A (ja) 2014-07-03
EP2251692A1 (fr) 2010-11-17
EP2508886A2 (fr) 2012-10-10
EP2009440A1 (fr) 2008-12-31
US20140094378A1 (en) 2014-04-03

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